Name: studying the activity of neuropeptides and other regulators of the excretory system in the adult mosquito
Uploaded: 2021-08-24T13:00:00
Description: Watch this Scientific Journal Video about Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito at JoVE.com

This research was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to AD and J-PP. AL and FS received NSERC CGS-M awards in support of their graduate research.

1. Making silicone-lined dishes
NOTE: This step should be done prior to the experiments. These dishes will be made to prepare the assay dish for dissections, and for contraction assay experiments.
<ol>
	<li>Prepare the silicone using a silicone elastomer kit following the manufacturer&#8217;s recommendations. Mix the two-part liquid components at a ratio of 10 to 1. Mix gently and thoroughly by inverting but be sure to minimize bubble formation.</li>
	<li>Pour the silicone elastomer into sta...

When ingesting a blood meal, haematophagus insects face the challenge of excess solutes and water in their haemolymph2. To cope with this, they have a specialized excretory system, which is tightly controlled by hormonal factors, allowing the insects to rapidly initiate post-prandial diuresis. The Ramsay assay and use of ion-selective microelectrodes allows for measurement of fluid secretion rates along with ion concentrations and transport rates in isolated insect MTs. Critical steps within these...

Maintenance of salt and water levels in insects allows them to succeed in many ecological and environmental niches, utilizing a variety of feeding strategies1. Most insects have evolved mechanisms to regulate the composition of their haemolymph within narrow limits in order to withstand the different challenges associated with their particular environment2. Terrestrial insects are often faced with the challenge of conserving water and the excretory system undergoes anti-diuresis to prevent loss of water and some essential salts, therefore, avoiding desiccation. In contrast, diuresis occurs when the insect feeds and is challenged with excess water and potentially salts3,4. Through their specialized and highly active excretory system, insects have evolved regulatory mechanisms acting to counter their osmoregulatory challenges. In adult Aedes aegypti mosquitoes, the excretory system is comprised of the Malpighian tubules (MTs) and hindgut, the latter of which is made up of the anterior ileum and posterior rectum5. MTs are responsible for generating primary urine, usually rich in NaCl and/or KCl. The primary urine is then modified through secretory and reabsorptive processes as it travels downstream of the tubule and enters the hindgut5. The final excreta can be hyper- or hypoosmotic to the haemolymph, depending on feeding/environmental conditions, and is enriched in toxic and nitrogenous wastes2.
MTs are ideal for studying many features of epithelial fluid and solute transport as they carry out a great variety of transport and excretory functions2,6. Through hormonal regulation2, MTs function by secreting ions and other solutes from the blood into the tubule lumen7, providing an osmotic gradient allowing water to be transported by aquaporins8,9, which collectively creates the primary urine, before traveling toward the reabsorptive hindgut2. Thus, by collecting the secreted fluid from isolated MTs, one can continuously monitor transepithelial transport of fluid and ions. Measuring secretion rate and urine composition provides insight on mechanisms responsible for transepithelial ion and fluid transport. A popular method for studying fluid secretion rates is the Ramsay assay, which was first introduced by Ramsay in 195310. In this method, the distal (closed) end of the tubule is treated with a hormone (or other test compound/drug), while the proximal (open) end is wrapped around a pin in water-saturated paraffin oil, which secretes the primary urine, accumulating as a droplet on the tip of the pin. Isolated MTs are able to survive and function for long periods (up to 24 h) under optimized in vitro conditions, which make them suitable and efficient models for fluid secretion measurement. Insects have open circulatory systems, thus the MTs are easily dissected and removed as they are usually freely floating in the haemolymph6. Additionally, with the exception of aphids&#8212;which lack MTs11&#8212;the number of MTs in a given insect species can vary considerably from four to hundreds (five in Aedes mosquitoes) allowing for multiple measurements from one insect.
The MTs in Aedes mosquitoes, in common with other endopterygote insects, are composed of two cell types forming a simple epithelium2,12; large principal cells, which facilitate active transport of cations (i.e., Na+ and K+) into the lumen, and thin stellate cells, which aid in transepithelial Cl- secretion13. The MTs are not innervated2, and instead are regulated by several hormones including both diuretic and anti-diuretic factors, allowing for the control of ion transport (mainly Na+, K+, and Cl-) and osmotically-obliged water2. Numerous studies have examined the hormonal regulation of Aedes MTs to understand the role of endocrine factors on transepithelial transport14,15,16,17,18. As shown in the representative results, the protocols herein demonstrate the effects of different hormonal factors on isolated MTs from adult female A. aegypti mosquitoes, including both diuretic and anti-diuretic control (Figure 1). The Ramsay assay is used to demonstrate how an anti-diuretic hormone, AedaeCAPA-1, inhibits fluid secretion of MTs stimulated by diuretic hormone 31 (DH31) (Figure 1).
The smaller size of insects has required the development of micro methods for measuring ionic activity and concentrations in fluid samples, or near the surface of isolated tissues such as the MTs and gut. Varying methods have been implemented, including the use of radioisotopes of ions19, which requires collection of the secreted fluid drops for measurement of ion concentrations20. Stimulated Aedes tubules in vitro typically secrete ~0.5 nL/min21, thus handling of such small volumes can pose a challenge and potentially introduce error upon transfer. As a result, ion-selective microelectrodes (ISMEs) have been extensively used to measure ion concentrations in secreted droplets of MTs in vitro. In this method, a reference electrode and ISME, filled with the appropriate backfill solution and ionophore, are positioned into the secreted urine droplet to determine ion concentrations22. Adapted from Donini and colleagues23, this current protocol uses a Na+-selective ionophore to measure ion activity in secreted droplets from stimulated MTs in adult Aedes mosquitoes. Since ion-selective microelectrodes measure ion activity, this data can be expressed as ion concentrations following the assumption that the calibration solutions and experimental samples share the same ion activity coefficient21 (Figure 1B,C).
The Scanning Ion-selective Electrode Technique (SIET) also makes use of ISMEs to measure ion concentration gradients in the unstirred layer adjacent to organs, tissues, or cells that are transporting ions. The ISMEs measure voltage gradients which can then be used to calculate the ion concentration gradients and direction and magnitude of ion flux across the organ, tissue, or cell20. In this technique, the ISME is mounted to a three axes manipulator controlled by computerized micro-stepper motors so that its 3D position is controlled to the micrometer level20. Voltages are measured at two points within the unstirred layer using a sampling protocol programmed into and controlled by computer software. The two points are typically separated by a distance of 20&#8211;100 &#181;m with one point within 5&#8211;10 &#181;m of the surface of the organ, tissue, or cell and the second point a further 20&#8211;100 &#181;m away. The difference in magnitude of voltages between the two points is calculated to obtain a voltage gradient24,25,26, which is then used to calculate the concentration gradient and subsequently the net flux using Fick&#8217;s Law24,27. This method is useful for assessing the transport of specific ions across different regions of the insect gut and MTs, or at specific timepoints following a bloodmeal or treatment exposure. For instance, the SIET can be used to understand how absorptive and secretory processes in the mosquito excretory system are regulated by hormones28 as well as different feeding behaviors and rearing conditions25. Previous work utilizing the SIET revealed sites involved in ion transport along the anal papillae and rectum of larval and adult mosquitoes24,28. The current protocol, described previously by Paluzzi and colleagues26, measures Na+ flux across the rectal pad epithelia of the adult female rectum (Figure 2).
The final segment of the mosquito excretory system requires coordinated muscular movement to help mix food and secrete waste26. Non-absorbable products of digestion from the midgut, along with primary urine secreted by the MTs, are passed through the pyloric valve and delivered to the hindgut2. Spontaneous hindgut contractions begin at the pyloric valve and occur in peristaltic waves, which are relayed over the ileum through the coordinated contraction of circular and longitudinal muscles surrounding the basal surface of epithelial cells26. Finally, the muscles within the rectum help to propel and eliminate waste through the anal canal. Although insect hindgut motility is myogenic, requiring extracellular Ca2+ to produce spontaneous contractions, these processes can also be regulated neuronally26,29,30. This exogenous regulation by the nervous system is important after feeding, as the animal must expel wastes from the gut and restore haemolymph balance31. As a result, performing in vitro bioassays to identify myostimulatory or myoinhibitory neuropeptides is useful in assessing how neurochemicals influence hindgut motility. The current protocol, performed by Lajevardi and Paluzzi28, uses video recordings to examine ileal motility in response to neuropeptides (Figure 3). Similarly, a force transducer or impedance converter may also be used to observe traces of contractions through a data acquisition software32,33. However, using video technology allows us to visually assess the organ and further analyze using a subset of parameters to identify the role of hormones on hindgut motility.
Using these techniques can help characterize factors that regulate and coordinate fluid and ion transport along the excretory system along with hindgut motility. Importantly, a functional link between the diuretic response by the MTs and hindgut motility is supported, as diuretic hormones, such as DH31 and 5HT, characterized by their ability to stimulate fluid secretion by the MTs, have also been found to exhibit myotropic actions along the mosquito hindgut21,34,35. These findings highlight the importance of stringent coordination between the MTs and hindgut during events such as post-prandial diuresis in insects requiring rapid waste elimination.
Herein, the detailed approach behind the Ramsay assay technique to measure fluid secretion rate in the mosquito, A. aegypti, and the use of ion-selective microelectrodes to determine Na+ concentrations within the secreted fluid of the MTs are described, which when combined allows for transepithelial ion transport rates to be determined. Additionally, the Scanning Ion-selective Electrode Technique and hindgut contraction assays are described to measure ion flux and motility, respectively, which helps to elucidate hormonal regulation of the hindgut (Figure 4).

<table>
	<tbody>
		<tr>
			<td>1 mL syringes</td>
			<td>Fisher Scientific</td>
			<td>14955456</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Corning Falcon (Fisher Scientific)</td>
			<td>C351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosillicate glass capillary filamented tubes 
			(OD 1 mm, ID 0.58 mm, length 100 mm)</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>used for ISME reference electrodes</td>
		</tr>
		<tr>
			<td>Borosillicate glass capillary filamented tubes 
			(OD 2 mm, ID 1.12 mm, length 102 mm)</td>
			<td>World Precision Instruments</td>
			<td>1B200F-4</td>
			<td>used for SIET reference electrodes</td>
		</tr>
		<tr>
			<td>Borosillicate glass capillary unfilamented tubes 
			(OD 1.5 mm, ID 1.12 mm, length 100 mm)</td>
			<td>World Precision Instruments</td>
			<td>TW150-4</td>
			<td>used for ISME and SIET electrodes</td>
		</tr>
		<tr>
			<td>CO2 pad</td>
			<td>Diamed</td>
			<td>GEN59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyltrimethylsilylamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>41716</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Custom</td>
			<td></td>
			<td>Can be fabricated by local machine shop</td>
		</tr>
		<tr>
			<td>Ferric chloride</td>
			<td>Sigma-Aldrich</td>
			<td>157740</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (Dumont #5)</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-748A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrated mineral oil</td>
			<td>Fisher Scientific</td>
			<td>8042-47-5</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>INFINITY1-2CB video camera</td>
			<td>Luminera</td>
			<td>INFINITY1-2CB</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulators (left and right handed)</td>
			<td>World Precision Instruments</td>
			<td>MMJL and MMJR</td>
			<td>Specific brand is not important so long as high quality manipulator</td>
		</tr>
		<tr>
			<td>Mineral Oil, Light</td>
			<td>Fisher Scientific</td>
			<td>0121-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien pins (0.1 mm stainless steel)</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-hardening modeling clay</td>
			<td>Sargent Art</td>
			<td></td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Olympus light microscope (FOR SIET)</td>
			<td>Olympus</td>
			<td>customized system</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur (transfer) pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution (0.1 mg/mL)</td>
			<td>Sigma-Aldrich</td>
			<td>A-005-M</td>
			<td>84 kDa</td>
		</tr>
		<tr>
			<td>Polyvinyl chloride (PVC)</td>
			<td>Sigma-Aldrich</td>
			<td>81395</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade</td>
			<td>Fine Science Tools</td>
			<td>10050-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10053-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#39;s Drosophila medium</td>
			<td>Sigma-Aldrich</td>
			<td>S0146</td>
			<td></td>
		</tr>
		<tr>
			<td>SIET system</td>
			<td>Applicable Electronics</td>
			<td>customized system</td>
			<td>Details available at: http://www.applicableelectronics.com/overview</td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>World Precision Instruments</td>
			<td>AGW1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ionophore II cocktail A</td>
			<td>Fluka</td>
			<td>99357</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard polystyrene Petri (culture) dishes</td>
			<td>Fisherbrand</td>
			<td>FB012921</td>
			<td>Any size would work, but 60 mm dishes are good for both dissections and assay</td>
		</tr>
		<tr>
			<td>Stereomicroscope with ocular micrometer</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutter P-97 Flaming Brown Pipette puller</td>
			<td>Sutter Instruments</td>
			<td>FGPN7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Chemical Company</td>
			<td>NC9285739</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>Sigma-Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR advanced hotplate stirrer - aluminum</td>
			<td>VWR</td>
			<td>9578</td>
			<td>Specific brand is not important</td>
		</tr>
	</tbody>
</table>

studying the activity of neuropeptides and other regulators of the excretory system in the adult mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

Application of DH31 against unstimulated MTs results in a significant increase in fluid secretion rate, confirming its role as a diuretic hormone in Aedes mosquitoes (Figure 1A). When tubules are treated with AedaeCAPA-1, a reduction in secretion rate is observed in DH31-stimulated MTs. Figure 1B demonstrates the use of ion-selective electrodes to measure Na+ concentrations in the secreted droplets. Treatment of...

Watch this Scientific Journal Video about Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito at JoVE.com

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

This protocol describes techniques that can be utilized by small animal physiologists to elucidate the role of neuropeptides and other hormonal factors that regulate the digestive and or excretory system. These techniques allow for measurements of micro size biological samples that would otherwise not be possible using techniques designed specifically for larger animal models, including, for example, rodents and teleus. This method can provide insight into the endocrine regulation of the gut, including transporting epithelial, as well as smooth muscle associated with the gastrointestinal tract in insects and in similarly sized non-insect species.Demonstrating the insect renal tubal procedures will be Farwa Sajadi, a PhD candidate from my laboratory. Additionally, a former graduate student from my lab Aryan Lajevardi will be demonstrating the hindgut focused protocols. Begin by preparing the Ramsay and contraction assays dishes for the experiments.Use pipette and fill wells with up to 20 microliters of solution. For unstimulated controls, fill the wells with 20 microliters of Aedes saline Schneider's medium. Once all wells are filled, pour hydrated mineral oil into the assay dish until the wells and Minutien pins are submerged.After immersing the tubule in the well, pick up the proximal end of the tubule with forceps, remove it from the bathing droplet, and wrap the end around the pin. Wrap the tubule around the pin twice keeping the length of tubule remaining in the bathing droplet consistent with the other tubules. To make a sodium selective microelectrode, use a one milliliter syringe to backfill the electrode with 100 millimolar sodium chloride.Ensure the backfill solution fills until the tip of the electrode. If air bubbles appear, gently flick the microelectrode or remove the solution and refill. Dip a 10 microliter pipette tip into the sodium selective ionophore solution.Line up the electrode perpendicular to the pipette, then place a gloved finger over the bottom of the tip to create pressure and expel a small drop of ionophore. Carefully touch the drop of ionophore to the microelectrode tip, making sure not to break it. Fill a small beaker halfway with 100 millimolar sodium chloride and place some modeling clay onto the inside at the top of the beaker.After the ionophore has been taken up, place the electrode tip down onto the wall of the beaker, letting the tips within the sodium chloride. Keep ISME in the beaker until it is ready to use. To make the ISME reference electrode, backfill an electrode with 500 millimolar potassium chloride and store it in a beaker.Coat the electrode tips using a solution of approximately 3.5%polyvinyl chloride, dissolved in tetrahydrofuran, to avoid displacement of the ionophore when submerged in paraffin oil. To calibrate the sodium electrode, place 10 microliter droplets of sodium chloride standard concentrations onto the edge of the Ramsey dish with the incubated Malpighian tubules. Place the standard droplets two centimeters apart with the higher concentration on top.Insert both the reference electrode and ion selective electrode over the chloride silver wires and fasten them securely using electrode holders that are attached to micro manipulators. Navigate both the electrodes toward the 200 millimolar sodium chloride droplet using the micro manipulators, ensuring that the electro tips do not touch the bottom of the dish. Turn on the electrometer to start recording and allow the reading to stabilize.Record the reading and continue to the next standard. After acquiring measurements of the fluid secretion rate, carefully move the reference and ion selective electrodes into the secreted droplet using the micro manipulators. Turn on the recording and allow the reading to stabilize, then record.Turn on the IPA-2 Ion/Polarographic Amplifier, the light microscope, and the computers. Place the sodium selective microelectrode on a holder consisting of a silver chloride wire and attach it into the female connector jack. Remove one reference electrode from the beaker with the potassium chloride, placing one finger at one end and tilting the glass capillary towards this finger to prevent the agar from falling out.Carefully place one end into the holder, ensuring that no bubbles form. If there is a bubble, remove the reference electrode, refill the holder with three molar potassium chloride and repeat. Place the electrode holder into the female connector jack.Following calibration, dissect the organ. Place the poly-L-lysine dish with the dissected sample on the microscope stage and insert the tip of the reference electrode inside the saline. Submerge the tip of the ion selective microelectrode in the saline, taking care not to break the tip.Use the manual adjustment knobs to adjust the microelectrode position while looking under the light microscope. Adjust the vertical position of the microelectrode such that its tip is on the same plane as the organ or tissue, then turn the motor switch to enable. Using the computer arrow keys, move the microelectrode horizontally to a position three millimeters away from the tissue to measure background recordings.When ready, begin recording by pressing F5, obtain five measurements of background activity. Move the microelectrode tip close to the tissue taking care not pierce the organ. Reduce the key hit sensitivity to place the microelectrode tip two micrometers directly to the right, perpendicular to the tissue.Obtain three recordings at site along the rectal pad to identify the site of greatest ion activity, then obtain baseline saline measurements at the site displaying greatest activity. To record hindgut contractions, fill one of the wells in the dish with a known volume of Aedes saline. Following dissection, carefully transfer the dissected hindgut attached to the mid gut into a well in another dish, making sure not to pinch the ileum.Submerge the gut in the saline inside the well and place Minutien pins into the mid gut and rectum. The ileum should not be under tension and spontaneous contractions originating at the pyloric valve at the anterior ileum should be observed. Connect the video camera to the stereoscopic microscope.Then place the dish containing the dissected organ under the microscope and record a video for two minutes. Application of DH31 against unstimulated Malpighian tubules results in a significant increase in fluids secretion rate, confirming its role as a diuretic hormone in Aedes mosquitoes. When tubules are treated with AedaeCAPA-1, a reduction in secretion rate is observed in DH31 stimulated Malpighian tubules.Ion selective electrodes were used to measure sodium concentrations in the secreted droplets. Treatment of DH31 on the MTs had no effect on the sodium concentration in the secreted droplet. However, with the application of AedaeCAPA-1, the sodium concentration in the secreted fluid was significantly increased.Additionally, compared with unstimulated controls, DH31 led to a significantly greater sodium transport rate, whereas AedaeCAPA-1 abolished this increase in DH31 stimulated tubules. The SIET was used to assess changes in sodium transport along the rectal pad epithelial of adult female mosquitoes. A leucokinin analog was used to examine changes in sodium absorption, which resulted in a four-fold decrease in sodium absorption compared to saline control.To assess the role of a neuropeptide pyrokinin2 on ileal motility, a Rhodnius prolixus analog was used, which was previously shown to activate the A.aegypti PK2 receptor enriched in the mosquito ileum. Relative to baseline levels, PK2 significantly inhibits ileal contractions. When performing a Ramsay assay with Malpighian tubules and measuring ion or fluid secretion rates, it is imperative that tubules aren't damaged during dissection or during the transfer into the assay dish.Following the Ramsay assay, specific membrane transporters can be targeted either pharmacologically or molecularly using reverse genetic techniques to discern their specific contribution to transepithelial transport of solutes in the simple epithelium.

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Research

JoVE Journal

Biology

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...